Oncolytic Adenovirus with SPAG9 shRNA Driven by DD3 Promoter Improved the Efficacy of Docetaxil for Prostate Cancer

Prostate cancer (PCa) is a common malignant tumor of the male urinary system and ranks the second in the causes of tumor-related deaths. Differential display code 3 (DD3) is a noncoding gene that is specifically expressed in PCa. High expression of sperm-associated antigen 9 (SPAG9) is closely related to tumorigenesis of PCa, and SPAG9 is a therapeutic target for PCa. In this study, a new oncolytic adenovirus DD3-ZD55-SPAG9 was constructed by using DD3 promoter to enhance the efficacy and safety of adenovirus. The combined use of DD3-ZD55-SPAG9 and docetaxel showed that DD3-ZD55-SPAG9 significantly improved the anti-tumor efficacy of docetaxel in PCa both in vitro and in vivo. The mechanism was related to the induction of tumor cell apoptosis and the inhibition of tumor cell invasion. In conclusion, DD3-ZD55-SPAG9 combined with docetaxel is an effective strategy for PCa therapy.


Introduction
Prostate cancer (PCa) is a common urinary system malignancy in men, ranking the second in the causes of cancerrelated deaths [1,2]. Due to the lack of early symptoms, most local or distant metastases already exist at the time of diagnosis. Relevant data showed that the 5-year survival rate of patients with early local PCa is close to 100%, but it will drop to below 30% for patients with distant metastasis [3]. Surgery and radiotherapy are the main treatment methods for early prostate cancer and achieve good therapeutic effects [4]. For advanced PCa, androgen deprivation therapy (ADT) is currently the main treatment method, including surgical castration or drug castration [5]. However, most patients will eventually develop castrationresistant prostate cancer (CRPC) with poor prognosis [6]. Docetaxel (DTX) is a semisynthetic broad-spectrum paclitaxel antitumor drug for a variety of cancers, including PCa, gastric cancer, liver cancer, breast cancer, and ovarian cancer. Docetaxel-(DTX-) based chemotherapy is the preferred treatment for CRPC in the guidelines [7]. How-ever, high-dose use of DTX will cause patients to have unbearable adverse reactions [8]. Therefore, it is necessary to find new drug combinations for the treatment of CRPC.
Oncolytic adenovirus can specifically infect, proliferate, and lyse tumor cells [9]. The virus released after cell lysis can spread to local or distant metastatic tumor tissues, producing amplification effect. The modified oncolytic adenovirus can provide multiple cloning sites for the insertion of therapeutic genes. Sperm-associated antigen 9 (SPAG9) gene encodes 766 amino acids and can participate in the MAPK signaling pathway through JNK structural homology to regulate cell activity [10]. SPAG9 is highly expressed in various malignant tumor tissues such as PCa, renal cancer, breast cancer, bladder cancer, and lung cancer [11][12][13][14][15]. Silencing the expression of SPAG9 in triple-negative breast cancer and liver cancer effectively inhibited the proliferation and migration of tumor cells [16,17]. Differential display code 3 (DD3) is a noncoding gene highly expressed in PCa tissues with little or no expression in normal prostate tissues or other tumor tissues [18]. The specificity and sensitivity of DD3 is higher than traditional PCa markers [19]. DD3 promoter is a PCa-specific promoter and restricts specific replication and propagation of oncolytic adenovirus in PCa, improving the safety and targeting of oncolytic adenovirus therapy.
In this study, we used DD3 promoter specificity to construct oncolytic adenovirus armed with SPAG9 shRNA to specially knockdown SPAG9 in PCa cells and examined whether constructed DD3-ZD55-SPAG9 improved the efficacy of DTX for PCa treatment in vivo and in vitro.

Statistical
Analysis. Data were expressed as mean ± SD and analyzed by SPSS 22.0 software. T-test was employed for the comparison between two groups. One-way ANOVA was employed for the comparison of multiple groups. P <0.05 was considered significant.

DD3-ZD55-SPAG9 Combined with DTX Inhibited PCa
Cell Proliferation. CCK-8 assay showed that DD3-ZD55-SPAG9 inhibited the proliferation of PC-3 and DU-145 cells in time-and concentration-dependent manner. After 48 hours of treatment with 10 MOI DD3-ZD55-SPAG9, the viability of PC-3 cells decreased significantly compared to PBS group (Figure 1(a)). DTX inhibited the proliferation of PC-3 in a time-and concentration-dependent manner (Figure 1(b)). Similar results were observed in DU-145 cells (Figures 1(c) and 1(d)). Furthermore, the combined use of DD3-ZD55-SPAG9 with DTX effectively inhibited the proliferation of PC-3 and DU-145 cells, with significant difference compared to other groups (Figures 1(e) and 1(f)).
To demonstrate that DD3-ZD55-SPAG9 has cytotoxicity only in prostate cancer cells, we performed CCK-8 assay on prostatic stromal myofibroblast WPMY-1 cells as the control. The results showed that the combined treatment of 5 MOI DD3-ZD55-SPAG9 and 1 ng/ml DTX had little cytotoxicity on WPMY-1 cells (Figure 1(g)).
3.6. DD3-ZD55-SPAG9 Combined with DTX Regulated the Expression of E-Cadherin, Vimentin, and MMP-2 Proteins in Xenograft Tumor. Immunohistochemical staining showed that the expression of SPAG9, vimentin, and MMP-2 was lower while the expression of E-cadherin was higher in each treatment group compared to the PBS group (Figure 6(a)). Quantitative analysis of optical density of SPAG9, E-cadherin, vimentin, and MMP-2 in DD3-ZD55-SPAG9+DTX, ZD55-SPAG9, DD3-ZD55-SPAG9, DTX, and PBS groups showed that the combined treatment group had the lowest SPAG9, vimentin, and MMP-2 protein expressions and the highest E-cadherin protein expression (Figures 6(b) and 6(c)). These results suggested that the combination therapy may inhibit tumor EMT in vivo.

Discussion
DD3 is one of the most prostate cancer-specific genes with high expression in prostate cancer tissues but no expression in prostate cancer adjacent tissues, benign prostatic hyperplasia, or normal prostate tissues [20]. Therefore, in this study, we constructed a recombinant oncolytic adenovirus DD3-ZD55-SPAG9 to specifically regulate the replication of adenovirus in PCa cells through DD3. Our results showed that DD3-ZD55-SPAG9 effectively silenced SPAG9; inhibited PCa cell proliferation, migration and invasion; and induced PCa cell apoptosis. Moreover, compared with ZD55-SPAG9, DD3-ZD55-SPAG9 exhibited significantly lower toxic effect on normal prostate cells. DTX is the first-line chemotherapy drug recommended for PCa, but it has toxicity and side effect on patients. In this study, we reduced the dosage of DTX and combined with DD3-ZD55-SPAG9 to explore antitumor effects in vitro and in vivo. The results showed that DD3-ZD55-SPAG9 +DTX had better effects than single treatment in inhibiting PCa cell proliferation, migration, and invasion and inducing PCa cell apoptosis.
EMT indicates the transformation of epithelial cells into mesenchymal cells and is one of the common mechanisms of tumor metastasis [21]. During EMT, there will be changes in the expression of various proteins, such as decreased expression of E-cadherin and increased expression of Ncadherin and Vimentin [22][23][24]. MMP family plays an important role in the process of tumor metastasis [25]. MMP-2 can digest tumor extracellular matrix, creating a pathway for tumor cell metastasis [26]. In this study we found that DD3-ZD55-SPAG9+DTX had better effects than single treatment in inhibiting SPAG9, vimentin, and MMP-2 expressions, and promoting E-cadherin expression in PCa in vitro and in vivo.
Promoting the apoptosis of tumor cells is one of the antitumor mechanisms of chemotherapy [27]. Consistently, in this study, we found that while DTX treatment alone promoted PCa cell apoptosis both in vitro and in vivo, the combined use of DD3-ZD55-SPAG9 and DTX significantly enhanced PCa cell apoptosis both in vitro and in vivo. These results suggest that the superior antitumor efficacy of DD3-ZD55-SPAG9 may be mediated by inducing PCa cell apoptosis.
In summary, we constructed oncolytic adenovirus DD3-ZD55-SPAG9 driven by DD3 promoter for targeted therapy of PCa. DD3-ZD55-SPAG9 exhibited superior efficacy and specificity to kill PCa cells both in vitro and in vivo without toxicity on normal prostate cells, which may be related to the induction of PCa cell apoptosis and the inhibition of EMT of PCa. DD3-ZD55-SPAG9 has promising application to enhance chemotherapy of PCa.

Data Availability
All data are included in this manuscript.

Conflicts of Interest
The authors declare that they have no conflicts of interest.